Methods for in vitro evaluating antimicrobial activity: A review

Mounyr Balouiri, Moulay Sadiki, Saad Koraichi Ibnsouda, Mounyr Balouiri, Moulay Sadiki, Saad Koraichi Ibnsouda

Abstract

In recent years, there has been a growing interest in researching and developing new antimicrobial agents from various sources to combat microbial resistance. Therefore, a greater attention has been paid to antimicrobial activity screening and evaluating methods. Several bioassays such as disk-diffusion, well diffusion and broth or agar dilution are well known and commonly used, but others such as flow cytofluorometric and bioluminescent methods are not widely used because they require specified equipment and further evaluation for reproducibility and standardization, even if they can provide rapid results of the antimicrobial agent's effects and a better understanding of their impact on the viability and cell damage inflicted to the tested microorganism. In this review article, an exhaustive list of in vitro antimicrobial susceptibility testing methods and detailed information on their advantages and limitations are reported.

Keywords: Agar diffusion method; Antimicrobial gradient method; Thin-layer chromatography (TLC)–bioautography; Time-kill test.

Figures

Fig. 1
Fig. 1
Agar diffusion methods: (A) disk-diffusion method of microbial extract using C. albicans as test microorganism, (B) agar well diffusion method of essential oil using Aspergillus niger as test microorganism, and (C) agar plug diffusion method of Bacillus sp. against C. albicans.
Fig. 2
Fig. 2
Broth microdilution method of plant extract against B. subtilis using resazurin as growth indicator.
Fig. 3
Fig. 3
0.5 McFarland microbial inoculum preparation by the direct colony suspension as recommended by CLSI guidelines.
Fig. 4
Fig. 4
Broth microdilution for antibacterial testing as recommended by CLSI protocol.

References

    1. Mayers D.L., Lerner S.A., Ouelette M. vol. 2. Springer Dordrecht Heidelberg; London: 2009. (Antimicrobial Drug Resistance C: Clinical and Epidemiological Aspects). pp. 681–1347.
    1. Guschin A., Ryzhikh P., Rumyantseva T. Treatment efficacy, treatment failures and selection of macrolide resistance in patients with high load of Mycoplasma genitalium during treatment of male urethritis with Josamycin. BMC Infect. Dis. 2015;15:1–7.
    1. Martin I., Sawatzky P., Liu G. Antimicrobial resistance to Neisseria gonorrhoeae in Canada: 2009–2013. Can. Commun. Dis. Rep. 2015;41:40–41.
    1. Berdy J. Bioactive microbial metabolites. J. Antibiot. 2005;58:1–26.
    1. Runyoro D.K., Matee M.I., Ngassapa O.D. Screening of Tanzanian medicinal plants for anti-Candida activity. BMC Complement. Altern. Med. 2006;6:11.
    1. Mabona U., Viljoen A., Shikanga E. Antimicrobial activity of Southern African medicinal plants with dermatological relevance: from an ethnopharmacological screening approach, to combination studies and the isolation of a bioactive compound. J. Ethnopharmacol. 2013;148:45–55.
    1. Nazzaro F., Fratianni F., De Martino L. Effect of essential oils on pathogenic bacteria. Pharmaceuticals. 2013;6:1451–1474.
    1. Heatley N.G. A method for the assay of penicillin. Biochem. J. 1944;38:61–65.
    1. CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests, Approved Standard, 7th ed., CLSI document M02-A11. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2012.
    1. CLSI, Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts, Approved Guideline. CLSI document M44-A. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 2004.
    1. Jorgensen J.H., Ferraro M.J. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin. Infect. Dis. 2009;49:1749–1755.
    1. Caron F. Antimicrobial susceptibility testing : a four facets tool for the clinician. J. Des. Anti-Infect. 2012;14 186-174.
    1. Nijs A., Cartuyvels R., Mewis A. Comparison and evaluation of Osiris and Sirscan 2000 antimicrobial susceptibility systems in the clinical microbiology laboratory. J. Clin. Microbiol. 2003;41:3627–3630.
    1. Kreger B.E., Craven D.E., McCabe W.R. Gram-negative bacteremia. IV. Re-evaluation of clinical features and treatment in 612 patients. Am. J. Med. 1980;68:344–355.
    1. Lopez-Oviedo E., Aller A.I., Martín C. Evaluation of disk diffusion method for determining posaconazole susceptibility of filamentous fungi : comparison with CLSI broth microdilution method. Antimicrob. Agents Chemother. 2006;50:1108–1111.
    1. Arikan S., Yurdakul P., Hascelik G. Comparison of two methods and three end points in determination of in vitro activity of Micafungin against Aspergillus spp. Antimicrob. Agents Chemother. 2003;47:2640–2643.
    1. Arikan S., Paetznick V., Rex J.H. Comparative evaluation of disk diffusion with microdilution assay in susceptibility testing of caspofungin against Aspergillus and Fusarium isolates. Antimicrob. Agents Chemother. 2002;46:3084–3087.
    1. CLSI, Method for Antifungal Disk Diffusion Susceptibility Testing of Nondermatophyte Filamentous Fungi, Approved guideline, CLSI document M51-A. Clincal and Laboratory Standards Institute, 950 West Valley Roead, Suite 2500, Wayne, Pennsylvania 19087, USA, 2010.
    1. Espinel-Ingroff A., Canton E., Fothergill A. Quality control guidelines for amphotericin B, itraconazole, posaconazole, and Voriconazole disk diffusion susceptibility tests with nonsupplemented Mueller–Hinton Agar (CLSI M51-A document) for nondermatophyte Filamentous Fungi. J. Clin. Microbiol. 2011;49:2568–2571.
    1. Fourati-Ben Fguira L., Fotso S., Ben Ameur-Mehdi R. Purification and structure elucidation of antifungal and antibacterial activities of newly isolated Streptomyces sp. strain US80. Res. Microbiol. 2005;156:341–347.
    1. Konaté K., Mavoungou J.F., Lepengué A.N. Antibacterial activity against β-lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: Fractional Inhibitory Concentration Index (FICI) determination. Ann. Clin. Microbiol. Antimicrob. 2012;11:18.
    1. De Billerbeck V.G. Huiles Essentielles et Bactéries Résistantes aux Antibiotiques. Phytotherapie. 2007;5:249–253.
    1. Das K., Tiwari R.K.S., Shrivastava D.K. Techniques for evaluation of medicinal plant products as antimicrobial agents: current methods and future trends. J. Med. Plants Res. 2010;4:104–111.
    1. Hausdorfer J., Sompek E., Allerberger F. E-test for susceptibility testing of Mycobacterium tuberculosis. Int. J. Tuberc. Lung Dis. 1998;2:751–755.
    1. Baker C.N., Stocker S.A., Culver D.H. Comparison of the E Test to agar dilution, broth microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria. J. Clin. Microbiol. 1991;29:533–538.
    1. Berghaus L.J., Giguère S., Guldbech K. Comparison of Etest, disk diffusion, and broth macrodilution for in vitro susceptibility testing of Rhodococcus equi. J. Clin. Microbiol. 2015;53:314–318.
    1. Gupta P., Khare V., Kumar D. Comparative evaluation of disc diffusion and E-test with broth micro-dilution in susceptibility testing of amphotericin B, voriconazole and caspofungin against clinical Aspergillus isolates. J. Clin. Diagn. Res. 2015;9:2013–2016.
    1. White R.L., Burgess D.S., Manduru M. Comparison of three different in vitro methods of detecting synergy : time-kill, checkerboard, and E test. Antimicrob. Agents Chemother. 1996;40:1914–1918.
    1. Denes É., Hidri N. Synergie et Antagonisme en Antibiothérapie. Antibiotiques. 2009;11:106–115.
    1. Gülmez D., Çakar A., Şener B. Comparison of different antimicrobial susceptibility testing methods for Stenotrophomonas maltophilia and results of synergy testing. J. Infect. Chemother. 2010;16:322–328.
    1. Bassolé I.H.N., Juliani H.R. Essential oils in combination and their antimicrobial properties. Molecules. 2012;17:3989–4006.
    1. Magaldi S., Mata-Essayag S., Hartung de Capriles C. Well diffusion for antifungal susceptibility testing. Int. J. Infect. Dis. 2004;8:39–45.
    1. Valgas C., De Souza S.M., Smânia E.F.A. Screening methods to determine antibacterial activity of natural products. Braz. J. Microbiol. 2007;38:369–380.
    1. Jiménez-Esquilín A.E., Roane T.M. Antifungal activities of actinomycete strains associated with high-altitude Sagebrush Rhizosphere. J. Ind. Microbiol. Biotechnol. 2005;32:378–381.
    1. Elleuch L., Shaaban M., Smaoui S. Bioactive secondary metabolites from a new terrestrial Streptomyces sp. TN262. Appl. Biochem. Biotechnol. 2010;162:579–593.
    1. Lertcanawanichakul M., Sawangnop S. A comparison of two methods used for measuring the antagonistic activity of Bacillus species. Walailak J. Sci. Tech. 2008;5:161–171.
    1. Ali-Shtayeh M.S., Ghdeib S.I. Abu. Antifungal activity of plant extracts against Dermatophytes. Mycoses. 1999;42:665–672.
    1. Mukherjee P.K., Raghu K. Effect of temperature on antagonistic and biocontrol potential of Trichoderma sp. on Sclerotium rolfsii. Mycopathologia. 1997;139:151–155.
    1. Kumar S.N., Nambisan B., Sundaresan A. Isolation and identification of antimicrobial secondary metabolites from Bacillus cereus associated with a Rhabditid Entomopathogenic Nematode. Ann. Microbiol. 2013;64:209–218.
    1. Goodall R.R., Levi A.A. A microchromatographic method for the detection and approximate determination of the different penicillins in a mixture. Nature. 1946;158:675.
    1. Fischer R., Lautner H. On the paper chromatographic detection of penicillin preparations. Arch. Pharm. 1961;294:1–7.
    1. Horváth G., Jámbor N., Végh A. Antimicrobial activity of essential oils: the possibilities of TLC-bioautography. Flavour Fragr. J. 2010;25:178–182.
    1. Mehrabani M., Kazemi A., Mousavi S.A. Ayatollahi. Evaluation of antifungal activities of Myrtus communis L. by bioautography method. Jundishapur J. Microbiol. 2013;6:1–7.
    1. Marston A. Thin-layer chromatography with biological detection in phytochemistry. J. Chromatogr. A. 2011;1218:2676–2683.
    1. Dewanjee S., Gangopadhyay M., Bhattabharya N. Bioautography and its scope in the field of natural product chemistry. J. Pharm. Anal. 2015;5:75–84.
    1. Choma I.M., Grzelak E.M. Bioautography detection in thin-layer chromatography. J. Chromatogr. A. 2011;1218:2684–2691.
    1. Grzelak E.M., Majer-Dziedzic B., Choma I.M. Development of a novel direct bioautography-thin-layer chromatography test: optimization of growth conditions for gram-negative bacteria, Escherichia coli. J. AOAC Int. 2011;94:1567–1572.
    1. Brantner A.H. Influence of various parameters on the evaluation of antibacterial compounds by the bioautographic TLC assay. Pharm. Pharmacol. Lett. 1997;7:152–154.
    1. Silva M.T.G., Simas S.M., Batista T.G. Studies on antimicrobial activity, in vitro, of Physalis angulata L. (Solanaceae) fraction and Physalin B bringing out the importance of assay determination. Mem. Inst. Oswaldo Cruz. 2005;100:779–782.
    1. Shahat A.A., El-Barouty G., Hassan R.A. Chemical composition and antimicrobial activities of the essential oil from the seeds of Enterolobium contortisiliquum (leguminosae) J. Environ. Sci. Health. B. 2008;43:519–525.
    1. Suleiman M., McGaw L., Naidoo V. Detection of antimicrobial compounds by bioautography of different extracts of leaves of selected South African tree species. Afr. J. Tradit. Complement. Altern. Med. 2010;7:64–78.
    1. Homans A., Fuchs A. Direct bioautography on thin-layer chromatograms as a method for detecting fungitoxic substances. J. Chromatogr. A. 1970;51:327–329.
    1. Hamburger M.O., Cordell G.A. A direct bioautographic TLC assay for compounds possessing antibacterial activity. J. Nat. Prod. 1987;50:19–22.
    1. Balouiri M., Bouhdid S., Harki E. Antifungal activity of Bacillus spp. isolated from Calotropis procera AIT. Rhizosphere against Candida albicans. Asian J. Pham. Clin. Res. 2015;8:213–217.
    1. Pfaller M.A., Sheehan D.J., Rex J.H. Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization. Clin. Microbiol. Rev. 2004;17:268–280.
    1. CLSI, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Approved Standard, 9th ed., CLSI document M07-A9. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2012
    1. Al-Bakri A.G., Afifi F.U. Evaluation of antimicrobial activity of selected plant extracts by rapid XTT colorimetry and bacterial enumeration. J. Microbiol. Methods. 2007;68:19–25.
    1. Liang H., Xing Y., Chen J. Antimicrobial activities of endophytic fungi isolated from Ophiopogon japonicus (Liliaceae) BMC Complement. Altern. Med. 2012;12:238.
    1. Monteiro M.C., de la Cruz M., Cantizani J. A new approach to drug discovery: high-throughput screening of microbial natural extracts against Aspergillus fumigatus using resazurin. J. Biomol. Screen. 2012;17:524–529.
    1. Kuhn D.M., Balkis M., Chandra J. Uses and limitations of the XTT assay in studies of Candida growth and metabolism. J. Clin. Microbiol. 2003;41:506–508.
    1. Reis R.S., Neves I., Lourenço S.L.S. Comparison of flow cytometric and alamar blue tests with the proportional method for testing susceptibility of Mycobacterium tuberculosis to Rifampin and Isoniazid. J. Clin. Microbiol. 2004;42:2247–2248.
    1. Ouedrhiri W., Bouhdid S., Balouiri M. Chemical composition of Citrus aurantium L. Leaves and zest essential oils, their antioxidant, antibacterial single and combined effects. J. Chem. Pharm. Res. 2015;7:78–84.
    1. Bouhdid S., Abrini J., Zhiri A. Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil. J. Appl. Microbiol. 2009;106:1558–1568.
    1. Castilho A.L., Caleffi-Ferracioli K.R., Canezin P.H. Detection of drug susceptibility in rapidly growing mycobacteria by Resazurin broth microdilution assay. J. Microbiol. Methods. 2015;111:119–121.
    1. Gehrt A., Peter J., Pizzo P.A. Effect of increasing inoculum sizes of pathogenic filamentous fungi on MICs of antifungal agents by Broth microdilution method. J. Clin. Microbiol. 1995;33:1302–1307.
    1. Meletiadis J., Meis J.F.G.M., Mouton J.W. Analysis of growth characteristics of filamentous fungi in different nutrient media. J. Clin. Microbiol. 2001;39:478–484.
    1. Gomez-Lopez A., Aberkane A., Petrikkou E. Analysis of the influence of tween concentration, inoculum size, assay medium, and reading time on susceptibility testing of Aspergillus spp. J. Clin. Microbiol. 2005;43:1251–1255.
    1. Rodriguez-Tudela J.L., Chryssanthou E., Petrikkou E. Interlaboratory evaluation of hematocytometer method of inoculum preparation for testing antifungal susceptibilities of filamentous fungi. J. Clin. Microbiol. 2003;41:5236–5237.
    1. CLSI, Reference Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved Standard, 2nd ed., NCCLS document M27-A2. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 2002.
    1. CLSI, Reference Method for Broth Dilution Antifungal Susceptibility Testing Filamentous Fungi, Approved Standard, 2nd ed., CLSI document M38-A2, 950 West Valley Roadn Suite 2500,Wayne, Pennsylvania 19087, USA, 2008.
    1. Arikan S. Current status of antifungal susceptibility testing methods. Med. Mycol. 2007;45:569–587.
    1. Lass-Flörl C., Cuenca-Estrella M., Denning D.W. Antifungal susceptibility testing in Aspergillus spp. according to EUCAST methodology. Med. Mycol. 2006;44:319–325.
    1. Petrikkou E., Rodri J.L., Gómez A. Inoculum standardization for antifungal susceptibility testing of filamentous fungi pathogenic for humans. J. Clin. Microbiol. 2001;39:1345–1347.
    1. Aberkane A., Cuenca-Estrella M., Gomez-Lopez A. Comparative evaluation of two different methods of inoculum preparation for antifungal susceptibility testing of filamentous fungi. J. Antimicrob. Chemother. 2002;50:719–722.
    1. CLSI, Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved Guideline, CLSI document M26-A. Clinical and Laboratory Standards Institute, 950 West Valley Roadn Suite 2500,Wayne, Pennsylvania 19087, USA, 1998.
    1. Cantón E., Pemán J., Viudes A. Minimum fungicidal concentrations of amphotericin B for bloodstream Candida species. Diagn. Microbiol. Infect. Dis. 2003;45:203–206.
    1. Espinel-Ingroff A., Fothergill A., Peter J. Testing conditions for determination of minimum fungicidal concentrations of new and established antifungal agents for Aspergillus spp.: NCCLS collaborative study. J. Clin. Microbiol. 2002;40:3204–3208.
    1. Espinel-Ingroff A., Chaturvedi V., Fothergill A. Optimal testing conditions for determining MICs and minimum fungicidal concentrations of new and established antifungal agents for uncommon molds: NCCLS collaborative study. J. Clin. Microbiol. 2002;40:3776–3781.
    1. CLSI, Methods for Antimicrobial Dilution and Disk Susceptibility of Infrequently Isolated or Fastidious Bacteria, Approved Guideline, 2nd. ed., CLSI document M45-A2. Clinical and Laboratory Standards Institute, 950 West Valley Roadn Suite 2500,Wayne, Pennsylvania 19087, USA, 2010.
    1. Menon T., Umamaheswari K., Kumarasamy N. Efficacy of fluconazole and itraconazole in the treatment of oral candidiasis in HIV patients. Acta Trop. 2001;80:151–154.
    1. Imhof A., Balajee S.A., Mar K.A. New methods to assess susceptibilities of Aspergillus isolates to caspofungin. J. Clin. Microbiol. 2003;41:5683–5688.
    1. Mock M., Monod M., Baudraz-Rosselet F. Tinea capitis dermatophytes: susceptibility to antifungal drugs tested in vitro and in vivo. Dermatology. 1998;197:361–367.
    1. Speeleveld E., Gordts B., Van Landuyt H.W. Susceptibility of clinical isolates of Fusarium to antifungal drugs. Mycoses. 1996;39:37–40.
    1. Clancy C.J., Huang H., Cheng S. Characterizing the effects of caspofungin on Candida albicans, Candida parapsilosis, and Candida glabrata isolates by simultaneous time-kill and postantifungal-effect experiments. Antimicrob. Agents Chemother. 2006;50:2569–2572.
    1. Klepser M.E., Ernst E.J., Lewis R.E. Influence of test conditions on antifungal time-kill curve results: proposal for standardized methods. Antimicrob. Agents Chemother. 1998;42:1207–1212.
    1. Crouch S.P., Kozlowski R., Slater K.J. The use of ATP Bioluminescence as a measure of cell proliferation and cytotoxicity. J. Immunol. Methods. 1993;160:81–88.
    1. Bozorg A., Gates I.D., Sen, Using A. Bacterial bioluminescence to evaluate the impact of biofilm on porous media hydraulic properties. J. Microbiol. Methods. 2015;109:84–92.
    1. Paloque L., Vidal N., Casanova M. A new, rapid and sensitive bioluminescence assay for drug screening on Leishmania. J. Microbiol. Methods. 2013;95:320–323.
    1. Finger S., Wiegand C., Buschmann H.J. Antibacterial properties of cyclodextrin–antiseptics–complexes determined by microplate laser nephelometry and ATP bioluminescence assay. Int. J. Pharm. 2013;452:188–193.
    1. Andreu N., Fletcher T., Krishnan N. Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence. J. Antimicrob. Chemother. 2012;67:404–414.
    1. Beckers B., Lang H.R., Schimke D. Evaluation of a bioluminescence assay for rapid antimicrobial susceptibility testing of mycobacteria. Eur. J. Clin. Microbiol. 1985;4:556–561.
    1. Finger S., Wiegand C., Buschmann H. Antimicrobial properties of cyclodextrin–antiseptics-complexes determined by microplate laser nephelometry and ATP bioluminescence assay. Int. J. Pharm. 2012;436:851–856.
    1. Galiger C., Brock M., Jouvion G. Assessment of efficacy of antifungals against Aspergillus fumigatus : value of real-time bioluminescence imaging. Antimicrob. Agents Chemother. 2013;57:3046–3059.
    1. Vojtek L., Dobes P., Buyukguzel E. Bioluminescent assay for evaluating antimicrobial activity in insect haemolymph. Eur. J. Entomol. 2014;111:335–340.
    1. Paparella A., Taccogna L., Aguzzi I. Flow cytometric assessment of the antimicrobial activity of essential oils against Listeria monocytogenes. Food Control. 2008;19:1174–1182.
    1. Ramani R., Chaturvedi V. Flow cytometry antifungal susceptibility testing of pathogenic yeasts other than Candida albicans and comparison with the NCCLS broth microdilution test. Antimicrob. Agents Chemother. 2000;44:2752–2758.
    1. Green L.J., Marder P., Mann L.L. LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability. Antimicrob. Agents Chemother. 1999;43:830–835.
    1. Green L., Petersen B., Steimel L. Rapid determination of antifungal activity by flow cytometry. J. Clin. Microbiol. 1994;32:1088–1091.
    1. Ramani R., Ramani A., Wong S.J. Rapid flow cytometric susceptibility testing of Candida albicans. J. Clin. Microbiol. 1997;35:2320–2324.
    1. Yousef A.E., Courtney P.D. Basics of stress adaptation and implications in new-generation foods. In: Yousef A.E., Juneja V.K., editors. Microbial Stress Adaptation and Food Safety. CRC Press; Washington DC: 2003. pp. 2–8.
    1. Tang Y.W., Stratton C.W. Springer; New York Heidelberg Dordrecht, London: 2013. Advanced Techniques in Diagnostic Microbiology, 2nd ed. pp. 937.

Source: PubMed

Patrocinadores e Colaboradores

Condições médicas

Intervenções de drogas

3
Se inscrever